Optical measuring of sample solutions is part of the standard procedures in, in particular, biotechnology or molecular biology. The analysis of the interactions between electromagnetic radiation and molecules or atoms in sample solutions, such as, for example, the transmission, reflection, absorption, fluorescence or scattering, allows a number of conclusions to be drawn about the compositions of the samples or the course of the biochemical processes.
In particular, the determination of a molar sample concentration of the sample solution is often performed in bioanalysis by measuring the extinction of monochromatic light of specific wavelengths. Under certain conditions the Lambert-Beer law, with which an unknown concentration can be determined either directly or by means of a calibration curve, applies here. In addition to the monochromatic light and constant external conditions, the presence of an ideal sample solution is a requirement for the application of the Lambert-Beer law. Only in the case of ideal sample solutions does the sample solution show the requisite linear dependency between the extinction and the concentration of the sample. Ideal sample solutions are concentrated so negligibly that between the dissolved molecules there cannot occur any interactions that could lead to non-linear dependencies. Under these conditions the measured extinction is proportional to the concentration and optical wavelength through the sample solution (layer thickness). An ideal sample solution can be produced by, for example, reducing too high a concentration of the sample by dilution. The range of the linear dependency can also be achieved by decreasing the layer thickness (for example, by using a flatter sample vessel) of a too highly concentrated sample until interactions can no longer occur.
Usually the layer thickness is determined by measuring a sample vessel, with which the sample solution is fed into the measuring device.
Sample vessels that are widely used include cuvettes, in which the sample solution is received in a measuring chamber between two optically transparent side walls. Such cuvettes are typically standardized, so that their receiving volume can be assigned a distance from the two optically transparent side walls and, as a result, a layer thickness of the measuring chamber. One drawback with such cuvettes is that the standardized volume content of >50 μl is usually too large for the very small sample amounts of <10 μl, which are customary in biotechnology or molecular biology. In addition, the volume content is fixed, so that it is not possible to change the layer thickness. Especially in the case of cuvettes that are intended for small sample volumes and have narrow and deep-lying measuring chambers, cleaning is barely possible. For this reason these cuvettes are usually designed as disposable articles and are not reusable.
The prior art also discloses reusable cuvettes for smaller sample amounts. The cuvettes, which are disclosed in the Offenlegungsschrift [published patent application] WO 2012/123395 A1 by the company HELLA GmbH and which are marketed under the name “TrayCell”, are suitable for sample volumes ranging from 0.7 to 10 μm. Compared to standard cuvettes, these cuvettes are very time-consuming to produce and, as a result, expensive.
Even in the case of cuvettes having different layer thicknesses the prior art has solutions. The Offenlegungsschrift DE 198 26 470 A1 discloses a cuvette, which is made of a synthetic plastic material and which comprises a measuring chamber having a rectangular cross section at a side ratio of preferably 5:1. The four side walls of the measuring chamber are optically transparent, so that the cuvette, rotated by 90 deg., can also be received in the measuring device. Owing to the rectangular cross section of the measuring chamber the sample can be easily measured in two layer thicknesses. The drawback is the large volume of the measuring chamber of ≥50 μl. The cuvette is made of a synthetic plastic material. Due to the lesser transparency for UV rays said cuvette does not lend itself very well to measurements with wavelengths of 220 nm. In addition, the cuvette is designed as a disposable article and cannot be reused again.
Another concept, in which small volumes of sample solutions can be measured in different layer thicknesses, is known from the German patent DE 10 2007 019 695 A1. In this case the invention relates to a chip cuvette in the form of a flat, planar support substrate, into which one or more measuring chambers and a channel system, connecting the measuring chambers, for receiving a sample volume are introduced. The channels and the measuring chambers are sealed with an optically transparent film. The measuring chambers of a chip cuvette can be configured so as to have different depths, so that a sample can be measured in different layer thicknesses. The chip cuvette is designed as a disposable article and cannot be reused.
In the aforementioned cuvettes the layer thickness of the sample solution is determined by the dimensioning of a measuring chamber, which is formed by a bottom and side walls and into which the sample liquid is filled. The measuring chamber forms the measuring volume respectively.
Another concept for a cuvette is known from the German patent DD 1077 83 B1. The described cuvette (called sample holder in this case) consists of two, essentially flat, transparent plates, which have surface structures on their surfaces that face each other; and their surface structures define the measuring surfaces (sample bearing regions in said patent). The two plates are arranged at a defined distance from each other, so that the measuring volumes, formed between the measuring surfaces, are limited laterally only by air. With respect to the measuring surfaces, they are designed in such a way that they are advantageously in the same plane as the other regions of the plate and are separated from said regions by ring-shaped grooves.
The plates are designed in such a way that they can be moved towards each other and have mechanical means for fixing their position with respect to each other. The cuvette is provided for extremely small measuring volumes of a few μl.
Even in the case of a device known from the Offenlegungsschrift WO 01/14855 A1, a small measuring volume of the sample solution of ≤10 μl in drop form is tensioned between two parallel optical surfaces, which are situated opposite each other, and is held in said drop form only by the surface tension of the liquid. In order to change the layer thickness of the sample solution, the distance between the optical surfaces can be varied in three positions by means of a controllable spacer, where in this case the drop can be compressed or pulled apart in accordance with the surface tension. Each of the opposing optical surfaces is formed as a raised surface on a leg, where in this case the two legs are connected to each other by means of a hinge at one of their two ends. The device can be folded open, so that the optical surfaces are freely accessible to receive the sample and are easy to clean. This feature makes the device reusable. This solution to the problem assumes greater complexity for driving and controlling the optical surfaces that can be moved towards each other. In addition, the different layer thicknesses can only be measured one after the other in succession, since the distance between the optical surfaces has to be changed between the measurements.
The aforementioned DE 20 2009 018 896 U1 discloses a cuvette comprising at least one measuring surface on each of the two legs (there arms), which are connected to each other by means of a swivel joint. When the cuvette is folded together, the legs are folded into a measuring position, in which the two measuring surfaces are situated opposite parallel to each other at a distance. The distance is suitable for holding a liquid sample between the measuring surfaces. In the closed state such a cuvette can be inserted into an optical measuring device in such a way that it crosses the light beam path of the measuring device; and the liquid sample is positioned in the light beam path. This patent discusses a wide variety of embodiments of a cuvette, in which the material and the geometry of the cuvette or also the surface of the measuring surfaces are varied. In each case, however, the measuring surface is a flat surface, so that the measuring surfaces are at the same distance from each other over the whole measuring range defined by said measuring surfaces. The distance between the two measuring surfaces can be set with a high degree of accuracy during the production of the cuvette, so that the cuvette is designed specifically for a thickness of the sample that is the result of the set distance. The cuvette may have one measuring range, but also a plurality of measuring ranges defined by two measuring surfaces.
The cuvettes (measuring plate cuvettes), disclosed in the aforementioned documents DD 1077 83 B1, WO 01/14855 A1 and DE 20 2009 018 896 U1, have in common that the liquid sample is held statically as a drop (drop volume) between two measuring surfaces by interfacial tension and adhesive force. The interfacial tensions denote the forces that act on the boundary between two different phases that are in contact with each other. That means that the two phases form a common interface that is under interfacial tension. In the case of the cuvettes disclosed in the aforementioned documents, there are in each case two interfaces between the drop and a glass surface and one interface between the drop and a gas, for example, air. The interfacial tension between a liquid and a gas is also referred to as the surface tension. A volume of a liquid, which is held together solely by the interfacial tension and adhesive force between the interfaces, constitutes a drop volume. A drop volume is in the range of 0.1 to 10 μl. Depending on the properties of the liquid and the surface finish of the measuring surfaces, the distance between the two measuring surfaces is limited to a range of 100 mm to 1.0 mm, so that a drop volume can be held between the two measuring surfaces.
In contrast to the aforesaid, larger volumes, which fill statically or dynamically a cuvette in the form of a container, for example, a box (box cuvette), follow the internal shape of the cuvette, for which reason there is no limit to be observed in the upward direction for dimensioning the distance between the measuring surfaces.